Established (tumor) cell line - or not...?

I've tried to establish some cancer cell lines from whole tumor using different protocols as described elsewhere.

Now I've finally managed to isolate some cells growing in RPMI, but how do I actually know that it is cancer cells and not benign cells from normal healthy tissue? Can non-malignant cells grow at all in plain RPMI without growth factors etc?

The answer lies in looking at the tumour and the cells you got out... for instance if your are growing a lung carcinoma, you would be looking to get lung cells out not fibroblasts, so look at the morphology. You could also profile the cells you got out, do they show the same histological types as the original tumour (e.g. receptor positivity such as estrogen receptor, etc.)?

For a primary culture I would expect several cell types to have grown, depending on the type of tumour, with the fastest growing out competing the rest. If this is the case, the fastest growing are probably fibroblasts and are likely not what you want. You may need to clone the tumour cells once you have identified the correct cell type.

Well, my established cells shows the right morphology. But are they malignant or not?

If you try to grow e.g. cells from normal healthy lung tissue, will they grow at all? Or is the fact that they are growing well suggesting that it's actually cancer cells? i started out with a piece of suggested tumor, but I think it's a possibility that this piece of tissue contains some normal cells as well. What if only the benign (normal) cells survive in RPMI?

Unfortunately, I don't have some specific markers to look for.

Maybe it's a stupid question, but what if a reviewer asks how I know that my isolated cells are tumor cells??? How do I prove that?

If you have normal and tumour cells in the culture, just keep them in culture for a little longer. The normal cells should die out and/or be out-competed by the tumour cells. You can verify that they are tumour cells by looking at profiles of gene expression and protein expression compared to normal cells from the same sample/patient.